The objectives of this grant are to better understand the basic immunobiology of costimulation and to design unique cancer immunotherapeutic strategies that invoke costimulation in new ways. The strategies advocated in this application are outgrowths of a decade-long effort by our investigative team to innovate protein transfer methods that can be used for costimulator "painting," as an empowering means for achieving quantitative and combinatorial costimulator neo-expression. Thus, protein transfer represents the driving concept and distinguishing feature of this line of investigation from its inception, and it is what sets it apart from the more widespread gene therapy-based strategies that are being applied elsewhere to cancer immunotherapy. Over the course of the current grant, both the protein transfer tools and the proteins transferred have evolved, and significantly, two protein transfer modalities emerged midway through the last funding period: 1) a simple two-component protein transfer method that uses palmitated-protein A:costimulator Fcgamma1 conjugates as "costimulator paints," and enables intratumoral, multi-costimulator vaccination; and 2) a new class of soluble "trans signal converter proteins (TSCP)," which in essence constitute "injectable paints." This renewal application offers two specific aims that leverage the first of these two new protein transfer modalities: 1) Analyzing immunobiological aspects of trans intercellular costimulation, with a focus on quantitative and synergistic effects of costimulation on T cell activation thresholds, and building upon this mode of costimulation for active cancer immunotherapy -- cellular cancer vaccination, via the intratumoral, combinatorial palmitated protein A:costimulator.Fcgamma1 protein transfer method that we innovated; and 2) Exploring immunobiological aspects of the cis auto-costimulation phenomenon that we discovered, with a focus on functional and molecular features of the activation state of costimulator-painted T cells, and building upon this mode of costimulation for passive cancer immunotherapy -- conferring anti-tumor immunity via the use of adoptively-transferred, costimulator-painted T cells.